Usefulness of
Polymerase Chain Reaction
in disease diagnosis
History
The
Polymerase Chain Reaction (PCR) was not a
discovery, but rather an invention
A special
DNA polymerase (Taq) is used to make many
copies of a short length of DNA (100-10,000 bp)
defined by primers
Kary
Mullis, the inventor of PCR, was awarded the
1993 Nobel Prize in Chemistry
Why to use PCR
PCR
can be used to make many copies of any
DNA that is supplied as a template
Starting
with one original copy an almost infinite
number of copies can be made using PCR
Amplified fragments of DNA can be sequenced,
cloned, probed or sized using electrophoresis
Defective
genes can be amplified to
diagnose any number of illnesses
Amplified
fragments can act as genetic
fingerprints
How PCR Works
PCR
is an artificial way of doing DNA
replication
Instead of replicating all the DNA present, only
a small segment is replicated, but this small
segment is replicated many times
As in replication, PCR involves:
Melting DNA
Priming
Polymerization
Basics of DNA replication
Origin of Replication
5
5
5
5
The Replication Fork
5
3
5
3
3
5
Primase
Laging Strand
Okazaki
fragment
3
RNA
Primers
Single
strand
binding
proteins
DNA
Polymerase
5
3
Helicase
Leading Strand
5
3
Functions And Their Associated
Enzymes
Function
Enzyme
Melting
DNA
Helicase
SSB
Proteins
Topisomerase
Polymerizing
Providing
DNA
primer
Joining nicks
DNA Polymerase
Primase
Ligase
Temperature
PCR
100
Melting
94oC
o
Extension 94 C
Annealing
Primers
50oC
50
72oC
Time
5
3
3
5
5
3
5
3
5
5
5
5
30x
Melting
3
3
Temperature
100
Melting
94oC
PCR
50
Time
Temperature
100
Melting
94oC
PCR
50
Time
3
Heat
5
Temperature
100
Melting
94oC
50
PCR
Annealing
Primers
50oC
Melting
o
Extension 94 C
72oC
Time
3
5
5
Temperature
PCR
100
Melting
94oC
50
Melting
94oC
Extension
Annealing
72oC
Primers
50oC
Time
3
Heat
5
Heat
5
5
30x
Temperature
100
Melting
94oC
50
PCR
Annealing
Primers
50oC
Melting
o
Extension 94 C
72oC
Time
3
5
5
30x
Temperature
100
50
0
3
Melting
94oC
PCR
Annealing
Primers
50oC
Melting
o
Extension 94 C
72oC
Time
5
5
5
Heat
5
Heat
5
30x
Temperature
PCR
100
50
0
3
Annealing
Primers
50oC
Melting
o
Extension 94 C
72oC
Time
5
5
5
Melting
94oC
30x
Temperature
100
Melting
94oC
50
0
3
Annealing
Primers
50oC
Melting
o
Extension 94 C
72oC
Time
5
5
PCR
Fragments of
defined length
30x
DNA Between The Primers Doubles
With Each Thermal Cycle
Number
1
2
16
32
64
Cycles
More Cycles = More DNA
Theoretical Yield Of PCR
Theoretical yield = 2n x y
Where y = the starting
number of copies and
n = the number of thermal cycles
If you start with 100 copies, at the end of 30
cycles?
2n x y
=230 x 100
=1,073,741,824 x 100
=107,374,182,400
How The Functions Of Replication
Are Achieved During PCR
Function
Melting
PCR
DNA
Polymerizing
DNA
Providing primer
Heat
Joining
N/A as
nicks
Taq
DNA
Polymerase
Primers
are
added to the
reaction mix
short
fragments are
Components of a PCR Reaction
Buffer
(containing Mg++ Cl ++)
Template DNA
2 Primers that flank the fragment of
DNA to be amplified
dNTPs
Taq DNA Polymerase (or another
thermally stable DNA polymerase)
Templates for PCR
Dried
blood
Semen stains
Vaginal swabs
Single hair
Fingernail scrapings
Insects in Amber
Egyptian mummies
Buccal Swab
Toothbrushes
Heat-stable polymerase
Thermus aquaticus:
Preparation of master mix
10
X PCR Buffer with MgCl2
Primers F
Primers R
dNTP (building blocks of DNA)
Taq polymerase
Template DNA (will vary for each sample)
Make up the volume with DW
The
reactions are carried out in thin walled
PCR tubes in a Thermal cycler
The amplified products
are
analysed
on
agarose
gels
and
visualized
on
Gel
documentation systems
Advantages of PCR
Quick
Reliable
Sensitive
Relatively
Specific
easy
PCR Applications in Pathology.
This technique is elegant and sensitive and has been
adopted quickly.
Diagnosis of infections is limited by the supply of
appropriate material for culture, protein analysis or
microscopy.
PCR has led to a better understanding of the pathobiology
of malignancy, allowing the analysis of mutations in
oncogenes and tumour suppressor genes.
PCR's greatest versatility is that it allows the examination
of formalin fixed paraffin wax embedded tissue.
PCR has brought significant progress in forensic pathology.
Disadvantage of PCR
Need for equipment
Taq polymerase is expensive
Contamination
False reactions
Internal control
Cross-reaction
Enrichment steps in (contaminated) samples
Skill building needed
Unspecific amplification
rtPCR
Nested primers
Nested primers
Multiplex-PCR
Multiple
primers are used in the same reaction mixture.
Simultaneously many genes can be targeted.
Produce amplicons of varying sizes that are specific to
different DNA sequences.
M
4
2
16sdys
paU
500bp
200bp
549bp
alr
361bp
sip
nuc
181bp
Hot start PCR.
It
reduces non-specific amplification
during the initial set up stages of the PCR.
The enzyme gets activated at the optimum
temperature.
It inhibits the polymerases activity at
ambient temperature, either by the binding
of an antibody.
Real Time PCR
Detection
and quantification of fluorescent reporter
the signal of which increases in direct proportion to
the amount of PCR product in a reaction.
Does not only measure the amount of end product
but its production in real time
The
identity of the product can further be
analysed by direct sequencing
gene <1..887 /gene="ompH" CDS <1..887
/gene="ompH" /codon_start=3 /product="outer
membrane protein" /protein_id="AAT37506.1"
/db_xref="GI:47681474" /"
1 gcgtttcatt caaagcatct catgatttag gcgaaggctt aagcgcatta gcttatacag
61 aacttcgttt tagtaaaaat gtacccgtgc aagtaaaaga ccaacaaggt gaagtagtac
121 gtgagtatga ggttgagaaa cttggtaaca atgttcacgt aaaacgtctt tatgcgggtt
181 tcgcgtatga aggtttaggt acattaacat tcggtaacca attaactatc ggtgatgatg
241 ttggtctatc tgactatacc tatttcaaca gtggtattaa taacctcctt tctagcggtg
301 aaaaagcaat taactttaaa tctgcagaat tcaatggttt cacatttggt ggtgcgtatg
361 tcttctctgc tgatgctgac aaacaagcat tacgtgatgg tcgcggtttc gttgtagcag
421 gtttatacaa cagaaaaatg ggtgatgttg gttttgcatt cgaagccggt tatagccaaa
481 aatatgtgaa acaagaagta gaacaagcac aagcaccaaa agtatttaaa gatgaaaaag
541 agaaagcttt catggtgggt gctgagttat catatgctgg tttagcgctt ggtgttgact
601 acgcacaatc taaagtgact aacgtagatg gtaaaaaacg tgctcttgaa gtgggtttaa
661 attatgacct taacgacaga gcgaaagttt acacagactt catctgggaa aaagaaggtc
721 ctaaaggtga tgttacaaga aaccgtactg tcgctgtagg ttttggttac aaacttcaca
781 aacaagtgga aacttttgtt gaagcagctt ggggtagaga gaaagactct gatggtgtaa
841 caacaaaaaa caacgtagta ggtacaggtt tacgcgtaca cttctaattt ttgttagaat
901 ctgaaaaaag ccagtgttaa acactggctt tttattgggt tttatttgtt ttacttacaa
961 taaattagga ttttgaaagt cgttacgcgg tcat
References
Mullis,
Kary (1990). The unusual origin of
the polymerase chain reaction. Scientific
American 262 (4): 56-61, 64-65
Polymerase
chain
reaction
(
http://en.wikipedia.org/wiki/Polymerase_ch
ain_reaction
)
